Ever since enzymes were first discovered, the question of how they achieve their remarkable catalytic proficiency has fascinated both chemists and biologists. This project aims to find general answers to this question for a particular class of enzymes: those that catalyze the transfer of a hydrogen ion, either a proton or a hydride, between parts of a substrate and between the enzyme and the substrate. Our focus on hydrogen stems from the fact that proton or hydride transfers occur as a part of nearly every enzymatic reaction, yet efficient transfer from or to an inactivated carbon or oxygen species is very difficult to accomplish non-enzymatically. For over two decades we have probed this question by a combination of protein crystallography, computational approaches, and site-directed mutagenesis. Along the way we have developed a number of techniques of general use in crystallography, structural biology, and drug discovery. Our studies have revealed the following general principles: (1) Enzymatic catalysis of proton transfer depends on perturbed pKa values for the groups involved. Consider a catalytic base. It appears that the basic nature of this group is increased, perhaps either by shielding it from the solvent or by placing an appropriately charged residue near it, although the mechanism of perturbation has not been proven in most cases. Enzymes also activate the substrate (i.e., increase the acidity of the carbon or oxygen acid) in two ways: by polarization, usually by hydrogen bond donation to a substrate oxygen atom, and by electrostatic stabilization of transition states. (2) Enzymatic catalysis of hydride transfer depends on proximity and orientation. Coenzyme strain does not appear to play a major role. Shielding of the substrate and/or cofactor from solvent does not seem to be essential, although it does occur sometimes. Polarization by, e.g., a metal ion can activate a substrate for hydride abstraction and donation, but often there is no obvious activation involved. For this renewal, we wish to focus on a set of questions that we believe have not been answered conclusively by any experiments or calculation. The questions are: (1) How are the catalytic acids and bases perturbed by the protein environment? (2) Do directed fluctuations (promoting vibrations) play a significant role in catalysis? To answer these questions we have selected several enzymes as model systems: four that catalyze proton transfer and three that catalyze both hydride and proton transfers. The specific methods we will employ include ultra-high resolution X-ray crystallography, neutron diffraction, combined QM/MM calculations, site-directed mutagenesis plus kinetic analysis, and a novel method for perturbing protein dynamics by binding ligands to sites remote from the active site. PUBLICE HEALTH RELEVANCE We are trying to understand how the environment of an enzyme makes difficult chemical reactions occur at blinding speed; such reactions are essential for every living cell, yet we don't understand all of the factors that go into producing this extraordinary chemical achievement. We have selected a particular class of reactions for study, and have devised a research plan that makes use of a large number of experimental and computational techniques to dissect what the protein is doing in each case to facilitate the chemistry. If we are successful, the principles we uncover could lead to the design of artificial enzymes for industrial and medical use.